The effect of hyaluronan on elastic fiber injury in vitro and elastase-induced airspace enlargement in vivo

This laboratory has previously described a method of preventing air-space enlargement in experimental pulmonary emphysema using aerosolized hyaluronan (HA). Although it was found that HA preferentially binds to elastic fibers (which undergo breakdown by elastases in emphysema), it remains to be shown that such attachment actually prevents damage to the fibers. In the current study, cell-free radiolabeled extracellular matrices, derived from rat pleural mesothelial cells, were used to test the ability of low molecular weight ( approximately 100 kDa) streptococcal HA to prevent elastolysis. Coating the matrices with HA significantly decreased elastolysis (P<0.05) induced by porcine pancreatic elastase (43%), human neutrophil elastase (53%), and human macrophage metalloelastase (80%). Concomitant in vivo studies examined the ability of an aerosol preparation of the streptococcal HA to prevent experimental emphysema induced by intratracheal administration of porcine pancreatic elastase. As seen with earlier studies involving bovine tracheal HA, a single aerosol exposure significantly decreased elastase-induced airspace enlargement, as measured by the mean linear intercept (107.5 vs 89.6 microm; P < 0. 05). Furthermore, repeated exposure to the HA aerosol for 1 month did not reveal any morphological changes in the lung. The results provide further evidence that aerosolized HA may be an effective means of preventing pulmonary emphysema and perhaps other lung diseases that involve elastic fiber injury.     

Synergistic effect of hydrogen peroxide and elastase on elastic fiber injury in vitro

This laboratory has previously shown that hyperoxia enhances airspace enlargement in a hamster model of elastase-induced emphysema. To further understand the mechanism responsible for this finding, the effect of oxidants on elastase activity was studied in vitro, using a radiolabeled elastic fiber matrix derived from rat pleural mesothelial cells. Matrix samples were treated with either 0.1%, 1%, 3%, or 10% hydrogen peroxide (H2O2) for 1 hr, then incubated with 1.0 microg/ml porcine pancreatic elastase for 2 hrs. Radioactivity released from the matrix was used as a measure of elastolysis. Results indicate that sequential exposure to H2O2 and elastase markedly enhanced elastolysis compared to enzyme treatment alone. A 22% increase in elastolysis was seen with 0.1% H2O2 (325 vs. 396 cpm; P < 0.05), whereas samples pretreated with 1%, 3%, and 10% H2O2 showed increases of 53% (274 vs. 420 cpm; P < 0.05), 71% (381 vs. 653 cpm; P < 0.01), and 38% (322 vs. 443 cpm; P < 0.01), respectively. Exposure to various concentrations of H2O2 alone (0.1% to 10%) produced only minimal elastolysis (<20 cpm). However, 1% H2O2 was capable of degrading peptide-free desmosine and isodesmosine, suggesting that exposure to this oxidant may reduce the stability of the elastic fiber matrix. With regard to lung diseases such as emphysema, H2O2 and other oxidants derived from inflammatory cells or the environment could possibly act as priming agents for elastase-mediated breakdown of elastic fibers, resulting in amplification of lung injury.     

Pulmonary fibroblasts: an in vitro model of emphysema. Regulation of elastin gene expression

Disruption and degradation of interstitial elastic fibers are significant characteristics of pulmonary emphysema. In order to examine the responses of elastogenic cells to the conditions mimicking degradation of interstitial pulmonary elastin, rat pulmonary fibroblast cultures were used as an in vitro model. Second passage fibroblasts were divided into two different environmental situations to represent cells adjacent to and remote from the site of elastase-digested matrix. One set of cell cultures was briefly digested with pancreatic elastase. The resultant digest was then added back incrementally to the medium of elastase-digested cell cultures and to the medium of a second set of undigested cultures. Both sets of cell cultures remained viable and metabolically active during these treatments (96 h of incubation) as judged by protein synthesis, cell number, and steady-state levels of beta-actin mRNA. However, the two sets of cultures exhibited opposite responses in elastin gene expression with addition of increasing amounts of the elastase digest. The elastase-digested cultures exhibited a 200% increase in extractable soluble elastin and a 186% increase in tropoelastin mRNA with the addition of increasing amounts of the elastase digest to the medium. Conversely, the amount of soluble elastin recovered from the undigested cultures decreased 75%, and the steady-state level of tropoelastin mRNA decreased 63%. Soluble elastin peptides generated from oxalic acid treatment of purified elastin were shown to decrease tropoelastin mRNA in undigested cell cultures in the same manner as the elastase digest. Based on these data, we propose that pulmonary fibroblast elastin gene expression can be controlled coordinately by the state of the extracellular matrix and solubilized peptides derived from that matrix. Such integrated regulation may serve to localize elastin repair mechanisms.     

Elastolytic activity of Pseudomonas aeruginosa elastase

Elastolysis of insoluble elastin by Pseudomonas aeruginosa elastase was found to be less specific (higher apparent Km value) but more active (higher activity) than with pancreatic elastase. Furthermore, pancreatic and P. aeruginosa elastases act synergistically during the initial stages of elastolysis. After extensive hydrolysis, the size distribution of digestion products was lower with P. aeruginosa than with pancreatic elastase. The higher extent of hydrolysis may be explained by the fact that, if pancreatic elastase needs at least six sub-sites for activity, P. aeruginosa elastase may hydrolyse tetrapeptides such as tetraalanine, or synthetic substrates such as furylacryloyltripeptides FA-X-Leu-Y, X and Y being Gly and/or Ala.     

Effects of elastase-induced emphysema on airway responsiveness to methacholine in rats

We examined the effects of elastase-induced emphysema on lung volumes, pulmonary mechanics, and airway responses to inhaled methacholine (MCh) of nine male Brown Norway rats. Measurements were made before and weekly for 4 wk after elastase in five rats. In four rats measurements were made before and at 3 wk after elastase; in these same animals the effects of changes in end-expiratory lung volume on the airway responses to MCh were evaluated before and after elastase. Airway responses were determined from peak pulmonary resistance (RL) calculated after 30-s aerosolizations of saline and doubling concentrations of MCh from 1 to 64 mg/ml. Porcine pancreatic elastase (1 IU/g) was administered intratracheally. Before elastase RL rose from 0.20 +/- 0.02 cmH2O.ml-1.s (mean +/- SE; n = 9) to 0.57 +/- 0.06 after MCh (64 mg/ml). A plateau was observed in the concentration-response curve. Static compliance and the maximum increase in RL (delta RL64) were significantly correlated (r = 0.799, P less than 0.01). Three weeks after elastase the maximal airway response to MCh was enhanced and no plateau was observed; delta RL64 was 0.78 +/- 0.07 cmH2O.ml-1.s, significantly higher than control delta RL64 (0.36 +/- 0.7, P less than 0.05). Before elastase, increase of end-expiratory lung volume to functional residual capacity + 1.56 ml (+/- 0.08 ml) significantly reduced RL at 64 mg MCh/ml from 0.62 +/- 0.05 cmH2O.ml-1.s to 0.50 +/- 0.03, P less than 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elastinolytic activity of horse leukocyte proteinases. Comparison with elastases from human leukocytes and porcine pancreas

Three proteinases from the azurophilic granules of horse leucocytes are typical elastases degrading elastin at neutral pH. Both proteinases: 1 and 2A exhibit similar elastinolytic activity, comparable with human leucocyte elastase (HLE). In relation to human enzyme, elastase 2B shows several-fold higher activity, which is comparable to the porcine pancreatic elastase activity (PPE). Similarly to HLE elastinolytic activity of the horse proteinases increases at higher ionic strength: twofold in case of 1 or 2A and fivefold for 2B. Significant activity observed during degradation of homologous lung elastin, implies the possible role of these enzymes during pathological injury of connective tissue in the lower respiratory tract and suggests similar pathogenesis of horse and human pulmonary emphysema.     

In vitro effects of elastase on rabbit craniomandibular joint articular disks.

This in vitro study correlates morphologic and radioimmunoassay (RIA) findings on the effects of elastase on the elastic fibers that are found in the rabbit craniomandibular joint (CMJ) articular disk. Articular disks were removed from rabbit CMJs at necropsy, and cut sagittally into two pieces which were incubated in 0.3 ml of phosphate-buffered saline containing either 0, 12.5, 25 or 50 units of porcine pancreatic elastase for either 1, 3 or 24 h. The quantitative RIA findings correlated well with the qualitative light-microscopic observations in that both methods showed a reduction in the amounts of elastin in the CMJ disks following enzyme treatment. However, the morphologic appearance of most of the elastase-treated disks suggested that the destruction of the elastic fibers was more extensive than was suggested by the results of the RIA which indicated that some elastin remained in the tissues of the disks even when the highest enzyme level and longest incubation period were combined. The results of this study also support the interpretation that the resorcin-fuchsin-stained fibers in the rabbit CMJ disk are elastic fibers.     

In vitro effects of elastase on periosteum of long bones: an histochemical, immunohistochemical and morphometric study

The aim of the study was to determine the in vitro effects of porcine pancreatic elastase on the periosteum of long bones and to what extent the effects are selective for the elastic fibres of the tissue. Twenty-eight new-born chicks' tibiae were incubated for 1 or 3 hours in different experimental conditions (PBS, 30 or 60 units (U)/ml of porcine pancreatic elastase) or immediately formalin fixed. The tibiae were then processed for histo-chemical (Verhoeff and van Gieson stain), immunohistochemical (anti-elastin antibody) and histomorphometric analysis. A decrease of periosteal elastic fibres in all the specimens incubated with elastase in comparison with non incubated specimens was evident. The effect of elastase was easily detectable even at the lower concentration (30 U/ml) and at the shorter time of incubation (1 h). The amount of elastic fibres decreased in accordance with the rise of enzyme levels and incubation time, while periosteal collagen fibre content was not substantially modified by elastase activity. Present data are a prerequisite to evaluate the in vitro and in vivo effects of experimental destruction of periosteal elastic fibres by elastase and to assess the role of these fibres in the growth process of long bones.     

Exercise intolerance and systemic manifestations of pulmonary emphysema in a mouse model

Background

Systemic effects of chronic obstructive pulmonary disease (COPD) significantly contribute to severity and mortality of the disease. We aimed to develop a COPD/emphysema model exhibiting systemic manifestations of the disease.

Methods

Female NMRI mice were treated 5 times intratracheally with porcine pancreatic elastase (emphysema) or phosphate-buffered saline (control). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, diaphragm dysfunction using isolated muscle strips, pulmonary hypertension by measuring right ventricular pressure, and neurohumoral activation by determining urinary norepinephrine concentration.

Results

Mean linear intercept was higher in emphysema (260.7 ± 26.8 μm) than in control lungs (24.7 ± 1.7 μm). Emphysema mice lost body weight, controls gained weight. Running distance was shorter in emphysema than in controls. Diaphragm muscle length was shorter in controls compared to emphysema. Fatigue tests of muscle strips revealed impaired relaxation in emphysema diaphragms. Maximum right ventricular pressure and norepinephrine were elevated in emphysema compared to controls. Linear correlations were observed between running distance changes and intercept, right ventricular weight, norepinephrine, and diaphragm length.

Conclusion

The elastase mouse model exhibited severe emphysema with consecutive exercise limitation, and neurohumoral activation. The model may deepen our understanding of systemic aspects of COPD.     

Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments.

Elastase-induced changes in flow were used to quantify the degradation of lung interstitial elastin. Degassed rabbit lungs were inflated with silicon rubber via airways and vessels. The lungs were cut into 1-cm-thick sections. Two chambers were bonded to each section to enclose the interstitium surrounding an arterial segment. Flow of albumin solution (0-5 g/dl) between the chambers was followed by that of the albumin solution with 0.25 g/dl pancreatic elastase solution. Driving pressure was 5 cmH(2)0, and mean interstitial pressure was either 0 or 10 cmH(2)O. Elastase caused an increase in flow in approximately 70% of the interstitial segments and a reduction in flow in the remaining segments. The elastase-induced response in flow was independent of both albumin concentration and mean interstitial pressure. Leukocyte elastase (5 units/dl) produced flow responses similar to those of 0.25 g/dl pancreatic elastase. The increased flow of leukocyte elastase was reduced by a subsequent flow with 0.25 g/dl pancreatic elastase but enhanced by a subsequent flow with a 10-fold lower concentration. A change in the order of the elastase flows reversed the concentration-dependent responses. This behavior suggests a complex interaction among the interstitial fibers after degradation by pancreatic and leukocyte elastase. Endogenous elastase-induced increases in interstitial permeability might affect blood-lymph barrier permeability, whereas elastase-induced cessation of flow might be related to the alveolar septal wall destruction observed in emphysema.     

The Receptor for Advanced Glycation End Products (RAGE) Contributes to the Progression of Emphysema in Mice

Several recent clinical studies have implied a role for the receptor for advanced glycation end products (RAGE) and its variants in chronic obstructive pulmonary disease (COPD). In this study we have defined a role for RAGE in the pathogenesis of emphysema in mice. RAGE deficient mice (RAGE-/-) exposed to chronic cigarette smoke were significantly protected from smoke induced emphysema as determined by airspace enlargement and had no significant reduction in lung tissue elastance when compared to their air exposed controls contrary to their wild type littermates. The progression of emphysema has been largely attributed to an increased inflammatory cell-mediated elastolysis. Acute cigarette smoke exposure in RAGE-/- mice revealed an impaired early recruitment of neutrophils, approximately a 6-fold decrease compared to wild type mice. Hence, impaired neutrophil recruitment with continued cigarette smoke exposure reduces elastolysis and consequent emphysema.     

Structural changes in elastic fibers after pancreatic elastase administration in hamsters.

Ultrastructural changes in lung parenchymal elastic fibers were studied morphometrically 1, 4, and 12 wk after a single 12-unit dose of pancreatic elastase and in a saline-instilled control group. The mean linear intercept of the parenchymal air spaces was increased in the 1-, 4-, and 12-wk post-elastase instillation groups compared with age-matched controls. The volume of alveolar connective tissue fibers predominantly composed of elastin (elastic fibers) was decreased by 35% 1 wk after the instillation of elastase but returned to control levels by 4 wk. Although the total volume of elastic fibers was normal 12 wk after instillation of elastase, the volume of elastic fibers in alveolar entrance rings remained significantly reduced. In serial sections of elastic fibers, numerous gaps or separations in the normally continuous band of elastic fibers that encircle each alveolus were identified 1 wk after elastase instillation. There were 169 +/- 8 (SE), 62 +/- 32, and 12 +/- 6 gaps per millimeter of alveolar entrance ring circumference at 1, 4, and 12 wk, respectively, in the elastase-treated groups. The number of gaps at 12 wk was equivalent to two gaps or discontinuities in the elastic fibers of every alveolar entrance ring. No gaps or separations in elastic fibers were detected at 1, 4, or 12 wk in the control groups. These defects occur in concordance with the progression of air space enlargement and presumably contribute to the progression of air space enlargement that occurs after the elastin content of the tissue has returned to normal.     

Hyaluronan blocks porcine pancreatic elastase-induced mucociliary dysfunction in allergic sheep.

Neutrophil elastase is a mediator common to asthma, chronic obstructive pulmonary disease, and cystic fibrosis and thought to contribute to the pathophysiology of these diseases. Previously, we found that inhaled hyaluronan blocked elastase-induced bronchoconstriction in allergic sheep through its control of tissue kallikrein. Here, we extend those studies by determining if inhaled hyaluronan can protect against the elastase-induced depression in tracheal mucus velocity, a surrogate marker of whole lung mucociliary clearance. We measured tracheal mucus velocity in allergic sheep before, and sequentially for 6 h after, aerosol challenge with porcine pancreatic elastase alone and after pretreatment with 1.5 or 6 mg aerosolized hyaluronan. Elastase (2.55 U) decreased tracheal mucus velocity. Pretreatment with 6 mg, but not 1.5 mg, hyaluronan inhibited the elastase-induced decrease in tracheal mucus velocity. Hyaluronan (6 mg) given 1 h after elastase challenge was ineffective, suggesting the involvement of secondary mediators. The elastase-induced depression in mucus transport appeared to be mediated, in part, by reactive oxygen species and bradykinin because pretreatment with either aerosolized catalase (38 mg/3 ml) or the bradykinin B2-receptor antagonist HOE140 (400 nM/kg) was also effective in blocking the response. These latter two findings are consistent with oxygen radical-induced degradation of hyaluronan with concomitant loss of its regulatory effect on tissue kallikrein, resulting in kinin generation. This hypothesis is supported by the demonstration that hyaluronan failed to block the oxygen radical-induced fall in tracheal mucus velocity resulting from xanthine-xanthine oxidase challenge and that inhaled bradykinin itself can slow mucociliary transport.     

Detection of antileukoprotease in connective tissue of the lung

An indirect immunofluorescence technique was applied to frozen sections of central and peripheral human lung tissue to search for extracellular localizations of antileukoprotease (ALP). Two monoclonal anti-ALP antibodies recognizing different epitopes and polyclonal anti-ALP antibodies were used. ALP was found to be localized along elastic fibers in alveolar septa, and also along elastic fibers in the walls of bronchi, bronchioles and blood vessels. Serous cells of bronchial submucosal glands showed labelling as well. In frozen sections of liver and spleen no label was found. Cells and elastic fibers were not labelled when lung tissue sections were processed with polyclonal or monoclonal anti-ALP antibodies, that were blocked with purified ALP before the immunostaining. The association of ALP with elastic fibers of human pulmonary connective tissue is of importance in understanding the role of the inhibitor in the defense of the lung parenchyma against the action of proteolytic enzymes, which is thought to result in emphysema.     

Thioredoxin prevents the development and progression of elastase-induced emphysema

Thioredoxin 1 (TRX1) is a redox (reduction/oxidation)-active protein that scavenges reactive oxygen species. Here we examined whether endogenous or exogenous administration of TRX1 prevented the development and progression of elastase-induced pulmonary emphysema. Mice were treated with intratracheal elastase via microspray on day 0, and were given recombinant human TRX1 (rhTRX1) every other day from days -1 to 21. To determine the effects of TRX1 on the progression of established emphysema, mice were treated intratracheally with elastase on day 0, and rhTRX1 was administered from days 14 to 21. Histopathologic examination was performed on day 21. TRX1-transgenic but not transgene-negative mice demonstrated a decrease in the physiological indicators of elastase-induced emphysema. TRX1 administration from days -1 to 19 significantly decreased the signs of elastase-induced emphysema. Moreover, TRX1 administration beginning 14 days after elastase treatment significantly slowed the progression of emphysema. TRX1 may be of clinical benefit for the treatment of COPD.     

烟雾加气管内滴入PPE 诱发非均质性肺气肿模型

目的:探讨烟雾与弹性蛋白酶(PPE)联合应用在兔阻塞性肺气肿模型形成过程中对肺组织X线、动脉血气、肺组织形态学结构的影响以及如何在较短时间内诱发出类似于人类疾病的非均质性肺气肿模型。方法:将40只小白兔随机分为吸烟组、注酶组、联合组及对照组4组,分别给予单纯香烟熏吸、气管内注入猪胰弹性蛋白酶、烟熏加气管内注入弹性蛋白酶及单纯气管内注入生理盐水作为对照组。7周后分别进行肺组织X线、动脉血气和肺组织形态学检查。结果:注酶组和联合组的平均肺泡数(Na)、肺泡隔面密度(Ds)及PaO2减少,PaCO2、肺总容积(TLC)和肺泡直径(Da)增加,与对照组比较,差异有显著性(P<0.05)。注酶组和联合组大鼠平均肺泡数(MAN)与正常对照组大鼠相比明显减少(P<0.05),平均肺泡面积(MAA)、平均内衬间隔(Lm)明显大于正常对照组(P<0.05),而吸烟组与正常对照组无统计学差异(P>0.05)。结论:烟雾可强化弹性蛋白酶在诱发肺气肿模型过程中的作用,二者联合应用可加快兔肺气肿模型的诱导过程。     

Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry.

Cutaneous aging and chronic exposure to UV irradiation leads to alterations in the appearance and biochemical composition of the skin. Members of the MMP family have been involved in the destruction of the extracellular matrix. Among them, gelatinases A and B were found to display elastolytic activity, in vitro. In this study, we first determined the ex vivo elastolytic potential of both endopeptidases, using human skin tissue sections and computerized morphometric analyses, and compared it with those of neutrophil elastase. In such conditions, gelatinase B (50 nM) induced 50% elastolysis. The percentage of elastic fibers degraded by gelatinase A (10-100 nM) never exceeded 10%. Elastolysis by gelatinase B and leukocyte elastase was characterized by a decrease in fiber length and an increase in the average diameter of the fibers. In addition, gelatinase B exhibited fibrillin-degrading activities. On the contrary, gelatinase A (50 nM) elicited up to 50% hydrolysis of collagen fibers, preferentially degrading type III collagen fibers. Gelatinase B did not promote any collagen degrading activity. Our data suggested that in vivo gelatinases could disrupt most extracellular matrix structures of human skin. Gelatinase B and to a much lesser extent, gelatinase A would degrade components of the elastic fibers network while gelatinase A, but not gelatinase B, would alter mostly collagen fibers and also degrade constituents of the dermo-epidermal junction.     

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats

ABSTRACT: BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg1 pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 mug) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1beta (IL-1beta), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1beta and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.     

Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling.

Enlargement of the respiratory air spaces is associated with the breakdown and reorganization of the connective tissue fiber network during the development of pulmonary emphysema. In this study, a mouse (C57BL/6) model of emphysema was developed by direct instillation of 1.2 IU of porcine pancreatic elastase (PPE) and compared with control mice treated with saline. The PPE treatment caused 95% alveolar enlargement (P = 0.001) associated with a 29% lower elastance along the quasi-static pressure-volume curves (P < 0.001). Respiratory mechanics were measured at several positive end-expiratory pressures in the closed-chest condition. The dynamic tissue elastance was 19% lower (P < 0.001), hysteresivity was 9% higher (P < 0.05), and harmonic distortion, a measure of collagen-related dynamic nonlinearity, was 33% higher in the PPE-treated group (P < 0.001). Whole lung hydroxyproline content, which represents the total collagen content, was 48% higher (P < 0.01), and alpha-elastin content was 13% lower (P = 0.16) in the PPE-treated group. There was no significant difference in airway resistance (P = 0.7). The failure stress at which isolated parenchymal tissues break during stretching was 40% lower in the PPE-treated mice (P = 0.002). These findings suggest that, after elastolytic injury, abnormal collagen remodeling may play a significant role in all aspects of lung functional changes and mechanical forces, leading to progressive emphysema.